POS0425 METABOLIC ALTERATIONS IN ACTIVATED FIBROBLAST-LIKE SYNOVIOCYTES FROM NON-INFLAMED SUBJECTS - MIMICKING EARLY STAGE OF RHEUMATOID ARTHRITIS
Authors: Y. Zhang, A. Nguyen, A. Rudin, C. Maglio
Journal: Annals of the Rheumatic Diseases (2022)

Background Proliferative cells, such as inflamed cells, depend on altered metabolic pathways to support their active proliferation. Synovial samples from patients with rheumatoid arthritis (RA) show reprogramming of different metabolic pathways such as glucose and glutamine metabolism [1]. However, it is unknown which metabolic pathways are altered in the early phases of RA pathogenesis, when non-inflamed fibroblast-like synoviocytes (FLS) are activated to a pro-inflammatory state. Our group has created an in-vitro cell model using FLS from subjects without inflammatory arthritis (non-inflamed FLS) after activation with adiponectin and tumour necrosis factor (TNF) to mimic the early stage of RA [2]. Objectives We aim to determine if the stimulation of non-inflamed FLS upregulates the expression of key-enzymes involved in glucose and glutamine metabolism and how the inhibition of those enzymes affects FLS activation. Methods FLS (passage 6-8) were isolated from synovial tissues of patients without inflammatory arthritis or osteoarthritis who underwent diagnostic arthroscopy due to a previous injury. FLS were cultured in DMEM medium (high glucose and GlutaMAX) containing 10% FBS and stimulated by known FLS-activators, i.e. TNF, interleukin 1 beta (IL-1 β), or adiponectin. For the inhibition experiments, cells were pre-treated with 25 mM 2-DG or 300 nM CB-839 for 4 hours before stimulation. Expressions of the enzymes were measured by western blot in whole-cell lysates and IL-6 was measured using ELISA in cell culture supernatants at 24 hours after stimulation. Cell proliferation was determined using MTT assay after 48 hours of stimulation. Results First, we compared the expression of hexokinase 2 (HK2), glutaminase C (GAC), and PFKFB3 in non-inflamed FLS before and after activation with adiponectin, TNF, and IL-1β. Expression of HK2 and GAC were upregulated in adiponectin- and TNF-activated FLS compared to unstimulated FLS. PFKFB3 was not affected by any of the stimuli. IL-1β did not affect the expression of the analysed enzymes (Figure 1A-C). As IL-1β did not affect the expression of metabolic enzymes, we continued stimulation only with adiponectin and TNF. TNF but not adiponectin significantly enhanced the proliferation of FLS without inhibition (Figure 1D). However, FLS proliferation was significantly reduced by pre-treatment with 2-DG, a glycolysis inhibitor, in unstimulated as well as TNF- and adiponectin-stimulated cells. CB-839, a glutaminase inhibitor, did not affect the proliferation of FLS (Figure 1D). Both TNF and adiponectin significantly upregulated the production of IL-6 in FLS. Pre-treatment with 2-DG significantly reduced the production of IL-6. CB-839 pre-treatment significantly reduced the production of IL-6 only in unstimulated FLS (Figure 1E). Figure 1. Metabolic alterations in non-inflamed FLS. Expression of hexokinase 2 (HK2; A), glutaminase C (GAC; B) and PFKFB3 (C) in non-inflamed FLS measured by western blot. Proliferation assay (MTT assay; D) and production of IL-6 (ELISA; E) was performed using cells treated with HK2 inhibitor 2-DG and glutaminase inhibitor CB-839. Statistical significance was determined as by one sample t test (A-C; n=7) or paired t test (D-E; n=4-6). Conclusion Our results show that the expression of key-enzymes regulating metabolic pathways can be enhanced by adiponectin and TNF in non-inflamed FLS. Moreover, we also show that inhibition of specific metabolic pathways can affect FLS activation differently depending on the cytokine stimulation. These results provide a deeper understanding of metabolic reprogramming in FLS in early RA. References [1]Bustamante, M.F., et al., Fibroblast-like synoviocyte metabolism in the pathogenesis of rheumatoid arthritis. Arthritis Res Ther, 2017. 19(1): p. 110. [2]Zhang, Y., et al., Recombinant Adiponectin Induces the Production of Pro-Inflammatory Chemokines and Cytokines in Circulating Mononuclear Cells and Fibroblast-Like Synoviocytes From Non-Inflamed Subjects. Front Immunol, 2020. 11: p. 569883. Disclosure of Interests None declared.